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Basic study on 6-iodo-D-m-Tyr
茨城県立医療大学紀要
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Original Article

Volume 24

Radioiodinated 6-iodo-D-meta-tyrosine: Characterization of Uptake in
DLD-1 Colon Cancer Cells and Biodistribution in Mice
Naoto Shikano1), Nobuyuki Yagi1), Ryuichi Nishii2), Kazuyo Ohe3), Syuichi Nakajima1), Masato Ogura1),
Ayako Ikeda1), Masato Kobayashi3), Naoto Yamaguchi4), Keiichi Kawai3)
1)

Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences
Diagnostic and Therapeutic Nuclear Medicine Team Department of Molecular Imaging and Theranostics, National Institute ofRadiological Science
3)
Division of Health Science, Graduate School of Health Sciences, Kanazawa University
4)
Center for Medical Science, Ibaraki Prefectural University of Health Sciences2)

Abstract
Introduction: 123I-6-iodo-D-meta-tyrosine (123I-6-D-mTyr) is an iodine-labeled D-amino acid employed potentially in molecularimaging of tumors. We used its 125I-labeled analogue (6-I-D-mTyr), which has a long half-life and is suitable for basic studies,
to examine the mechanism of 123I-6-D-mTyr accumulation in DLD-1 human colon cancer cells and its biodistribution in normal
mice.
Methods: DLD-1 cells (5.0×105) suspended in 5 ml of culture medium (Dulbecco`s Modified Eagles Medium) containing fetal
bovine serum and L-glutamine were seeded into 6-cmφ culture dishes and cultured at 37°C, CO2 5%, and pH 7.4. Time curves
for the uptake of 6-I-D-mTyr (18.5 kBq) into DLD-1 cells were constructed, and the transport systems were analyzed by uptakeinhibition experiments using 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a system L–specific inhibitor) and2-(methylamino)isobutyric acid (a system A–specific inhibitor) at an uptake temperature of 4°C. Also, 6-I-D-mTyr (11.1 kBq)
was administered to male ddY mice (6 weeks old) by injection into the tail vein, and the time-course of its biodistribution wasdetermined.
Results: The uptake of 6-I-D-mTyr into DLD-1 cells peaked at 10 min. Membrane transport inhibition studies indicated that ca.
70% of the incorporated amount was due to membrane transport (ca. 50% was due to transport system L, and the other ca. 20%
was due to membrane transport via an unknown mechanism), whereas inhibition unsaturated uptake was estimated to be ca. 30%(4°C). Physiologic accumulation of 6-I-D-mTyr in normal mouse tissues was low overall, and clearance from the blood wasobserved to be rapid. Accumulation in the stomach was also low, indicating excellent metabolic resistance to de-iodination
enzymes.
Conclusions: In normal mice, the concentration of 6-I-D-mTyr in organs and tissues decreased rapidly following administration,
whereas in DLD-1 mice, both the uptake and retention of 6-I-D-mTyr were excellent. These results highlight the potential of
6-I-D-mTyr as a new labeling agent for use in molecular imaging of tumors.
Key words: 6-iodo-D-meta-tyrosine, system L, neutral amino acid, DLD-1 cells

Address: Naoto Shikano. Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi,
Inashiki-gun, Ibaraki 300-0394, Japan
Phone: +81-29-840-2217, Fax: +81-29-840-2317, E-mail address: sikano@ipu.ac.jp

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茨城県立医療大学紀要

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1. Introduction

第 24 巻

tumor imaging.7) Also, it was reported that the D-isomers
for a number of AAs, including O-11C-methyl tyrosine,

It is well known that, depending on the optical isomer,

O-18F-fluoromethyl tyrosine, O-18F-fluoroethyl tyrosine,

the biological affinity of drugs often differs significantly.

O-18F-fluoropropyl tyrosine, and 11C-methionine, showed

The same can be said in regard to the optical isomers oflower uptake by tumors than the L-isomers, but theirnatural and non-natural amino acids (AAs). D-AAs aretumor/blood and tumor/normal tissue ratios (that is, thenot common compared with L-AAs. For that reason,

contrast between the tumors and the background) weremuch remains unclear regarding the affinity of D-AAs andsuperior.8,9) An in vitro study found that, compared with

their analogues for intermediates of biological pathways,

O-18F-L-fluoroethyl tyrosine, the D-isomer accumulated

and recent years have seen an upsurge in research on

only minimally in human colon cancer cells,10) and the

D-AAs in the field of radiopharmaceuticals.

D-isomer also showed almost no accumulation in mouse

Our first attempt to use a D-AA as a diagnostic

brain.11) Schober et al.12) reported similar accumulation ofradiopharmaceutical was reported by our co-authors,

L- and D-11C-methionine in a malignant glioma, whereas

Kawai et al., who developedI-3-iodo-D-tyrosine

Bergström et al.13) found that accumulation of D-11C-(3-125I-Tyr) as a diagnostic agent for amino acid membrane

methionine into brain tumor was lower than that of L-11C-

transport in the pancreas.1) Langen et al. reported thatmethionine. More recently, our basic study found that

accumulation of the D-isomer of cis-4-[ F]fluoro-proline

D-3H-methionine accumulated in human-derived cultured

in the human brain parenchyma was 5 times greater thantumor cells as result of transport by systems L and ASC,

that of the L-isomer, an interesting finding that at firstwhereas L-3H-methionine was transported by system L; inglance would seem to go against expectation.2)

mice bearing transplanted tumors, D-3H-methionine

125

18

From the standpoint of using the D-isomers of natural
AAs, it was reported that with 14C-methionine3) andaccumulation was significantly higher than that of L-14Cmethionine.14)

C-leucine4), the D-isomers are more highly accumulated

We carried out a series of studies using radioactive

than L-isomers in the pancreas and transplanted tumor

iodine–labeled compounds, including meta-tyrosine, withcells. Also, we investigated the uptake of a number ofthe ultimate objective of developing a diagnostic imaging

natural AAs and their respective optical isomers inagent for use in positron emission tomography (PET) andChinese hamster ovary-K1 (CHO-K1) cells. We foundsingle-photon

that

C-D-

(SPECT).5,14–18) In the present study, we focused on 125I-6-

C-D-leucine and their L-isomers

iodo-D-meta-tyrosine (6-I-D-mTyr) (Fig. 1), the D-isomeraccumulated at comparably high levels in cells, and theof iodine-labeled meta-tyrosine. We investigated theL-isomers

C-tyrosine,

accumulation of 6-I-D-mTyr in DLD-1 colon cancer

C-arginine showed high stereo-

cells19) and the expression of AA transporters and

14

5)

C-D-phenylalanine,14

methionine, andof

C-D-alanine,

14

14

C-serine,

14

C-glutamine, and

14

14

14

C-valine,14

14

selectivity in regard to accumulation in CHO-K1 cells.5)

emissioncomputed

tomography

determined the biodistribution in mice.

There have also been a number of reports regarding theIuptake of non-natural D-AAs in tumors. In tumor-bearing
animals, minimal difference was observed with respect tothe accumulation of 2-iodo-D-phenylalanine and its
L-isomer according to the optical isomer in normal tissues,
but 2-iodo-D-phenylalanine’s accumulation in tumors
was lower than that of the L-isomer.6) However, a study in

HO

H

NH2
COOH

Fig. 1. Structure of 6-iodo-D-meta-tyrosine (6-I-D-mTyr).

normal humans found that, compared with the L-isomer,
the D-isomer was rapidly cleared from the blood andexcreted in the urine, indicating greater suitability for

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Basic study on 6-iodo-D-m-Tyr

2. Materials and Methods

3

used for experiments on the 3rd or 4th day after inoculation.5)
Cell proliferation curves were prepared to determine theduration of the logarithmic growth phase.

2.1. Materials
The radiolabel precursor (D,L-mTyr) was purchasedfrom Sigma-Aldrich Japan Co. 125I-NaI was purchased

2.4. Time Course of 6-125I-D-mTyr Uptake

from Amersham Pharmacia Biotech. D-[14C(U)] tyrosine

When DLD-1 cells had been cultured to a density of

was purchased from American Radiolabeled Chemicals

about 80% confluence (3-4 days after inoculation), theInc. DLD-1 (human colon adenocarcinoma, JCRB9094)

medium was replaced with 5 ml of phosphate-bufferedcells were purchased from JCRB. Plastic tissue culture

saline (PBS) containing Na+ (37°C, pH 7.4). The cells

dishes (diameter 6 cmφ, Cat. no. 150288) and plastic

were then incubated at 37°C for 10 min. Next, the PBS

culture flasks (surface area 25 cm , Cat. no. 163371) were

was replaced with 2.0 ml of uptake solution containing

purchased from Nalge Nunc International. 2-Amino-

18.5 kBq of 6-I-D-mTyr. The incubation was conductedbicyclo[2,2,1]heptane-2-carboxylic acid (BCH, a systemusing PBS with Na+ uptake solution (NaCl, 137 mM; KCl,

L–specific inhibitor), 2-(methylamino)isobutyric acid

3.7 mM; Na2HPO4, 8.0 mM; KH2PO4, 1.5 mM;(MeAIB, a system A–specific inhibitor), natural L/D-

CaCl2·2H2O, 1.8 mM; MgCl·6H2O, 1 mM). The cells

amino acids, and other chemicals of reagent grade were

were then incubated for 5, 10, 15, 30, and 60 min. Next,

purchased from Kanto Chemical Co., Inc.

the medium was removed, and the cells were then washed2

twice using 5.0 ml of cold PBS and solubilized using 2.0
ml of NaOH (2 N). The radioactivity of each aliquot (5002.2. Labeled Amino acids
We separated L-mTyr from D,L-mTyr using high-ml) was counted using an auto-well counter (Aloka,

performance liquid chromatography (HPLC) with aJapan; ARC-380). The result for each set of experimental

Crownpak CR(+) chiral column (Daicel Chemical

conditions was the mean of 4 dishes.

Industries, Ltd.). Separated L-mTyr was labeled with
I-NaI (8.1×1019 [Bq/mol]) and Chloramine-T (Kanto

125

2.5. Labeled Amino Acid Uptake Inhibition Study

Chemical Co., Inc.). Radioiodination of D-mTyr produces

In the analysis of transport system L, we removed the16)

2 major geometric isomers: 6-iodo- and 4-iodo-D-mTyr.

culture fluid from the culture dishes, added 5 ml of 37°C

To separate and purify these 2 isomers, we performed

PBS, and incubated the dishes for 10 min at 37°C, 5%

HPLC with a Nova-Pak C18 column (Waters Co.).

CO2, and pH 7.4. The PBS was then removed. To

Radiochemical yield was checked using a silica gel thin-

investigate the concentration dependence of uptake, welayer chromatography kit (Merck). The labeled compounds

added 0.1, 0.5, 1.0, 5.0, and 10.0 mM BCH as a specificwere used in their noncarrier-added condition.

transport system inhibitor to 5 ml of 37°C Na+-free PBS
containing 18.5 kBq of carrier-free 6-I-D-mTyr (137 mM
choline-Cl, 3.7 mM KCl, 8.0 mM K2HPO4, 1.5 mM

2.3. Cell Culture and Proliferation Curves
DLD-1 cells (10 cells/ml) were incubated in 6-cm

KH2PO4, 1.8 mM CaCl2･2H2O, 1 mM MgCl·6H2O) as andishes with 5 ml of Dulbecco’s modified Eagle’s medium

uptake solution. The dishes were again incubated for 10(Cat. no. D5796, Sigma Chemical Co., St. Louis, MO,

min under the conditions described above. The uptake

USA) containing 10% fetal calf serum at 37°C in a 5%

solution was removed, and the cell surface was rinsed two

CO2 atmosphere (pH 7.4). Sub-culturing was performed

times with 5 ml of ice-cold PBS. In addition, 2 ml of

every 5 days using 0.02% ethylenediamine tetraacetic

NaOH (2 N) was added, and the dishes were left standingacid and 0.05% trypsin. DLD-1 cells were seeded on 60-

at room temperature for 24 h to solubilize the cells. The

mm dishes at a density of 5×10 cells in 5.0 ml of theradioactivity in an aliquot (500 µl) of each sample wascomplete culture medium. The growth curve was plottedmeasured.

5

5)

5

based on growth up to 6 days after inoculation. Cells were

In the analysis of transport system A, PBS with Na+ was

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茨城県立医療大学紀要4

第 24 巻

used as the uptake solution, with the specific transport

Primers (Table 1) were synthesized by Nihon Genesystem inhibitor MeAIB at 0.1, 0.5, 1.0, 5.0, and 10.0 mM

Research Laboratories, Inc. (Miyagi, Japan). The artificial

to examine the concentration dependence of uptake. The

plasmid used for the calibration curve was synthesized by

specific

concentration

GenScript (Piscataway, NJ, USA). The qRT-PCR reagent

dependence was determined based on the radioactivity

used in this study was Brilliant II Fast SYBR Green qRT-

levels recorded in the above experiments, and analysis of

PCR Master Mix (Stratagene Products Division, Agilent

the transport system was carried out at the concentration

Technologies). The thermal profile of the reaction was asshowing the greatest inhibitory effect.

follows: 2 min at 95°C for 1 cycle, 5 s at 95°C followedtransportsysteminhibitor

We examined the system L specificity of transport ofby 20 s at 60°C for 40 cycles, 1 min at 95°C followed by

the labeled amino acids in DLD-1 cells. One of the

30 s at 55°C and 30 s at 95°C for 1 cycle. In addition,

following compounds was added to cell cultures at a final

serial dilutions of artificial plasmids were performed. The

concentration of 10 mM or 1 mM: BCH in incubation

samples used were created using a dilution series ranging

medium without Na+; or 1 mM MeAIB in incubationfrom 1×102 to 1×108 copies per tube for the calibration

medium with Na+. The cells were then incubated in 2 ml

curve. All reactions were performed in triplicate.

of incubation medium with/without Na containing 18.5+

Table 1. Primers for real-time PCR.

kBq of 6-I-D-mTyr or D-[14C(U)] tyrosine for 10 min at
37°C. In the medium without Na+, NaCl was replacedwith the same concentration of choline-Cl. To examine
effects of low temperature, we incubated cells for 10 min

4F2hc

at 4°C. We measured the radioactivity associated withsolubilized cells using an ARC-380 well-type scintillation
counter (Aloka) for

I-labeled compounds and an

125

LAT1

LS6500 liquid scintillation counter (Beckman Instruments,
Fullerton, CA, USA) for 14C-labeled compounds.

LAT2

2.6. qRT-PCR Analysis of DLD-1 Cells
Total RNA was harvested from each tumor cell using
an RNeasyb Mini kit (Qiagen K. K, Tokyo, Japan). The

LAT3

quality of the total RNA was evaluated using a bioanalyzer(Agilent

Technologies,

Santa

Clara,

CA,

USA).

Complementary DNA (cDNA) was amplified only from

LAT4

high-quality total RNA using an AffinityScript qRT-PCR
cDNA Synthesis kit (Agilent Technologies). The thermal
profile of the reaction was as follows: 5 min at 25°C for 1

ACTB

cycle, 15 min at 42°C for 1 cycle, and 55 min at 95°C for
1 cycle. The genes analyzed by qRT-PCR were Na+independent transport L systems (LAT1, LAT2, LAT3,GAPDHPrimerSequence (5’-3’)

Forwardtaccggggtgagaactcgt

Reversecagccaaaactccagagcat

Forwardgtggaaaaacaagcccaagt

Reversegcatgagcttctgacacagg

Forwardttgccaatgtcgcttatgtc

Reverseggagcttctctccaaaagtcac

Forwardtttggtggcatctgcctaa

Reverseattaacgtggagcgcaggt

Forwardcagggagaccctctgtgg

Reversecggtagcagatcaggtagagc

Forwardccaaccgcgagaagatga

Reverseccagaggcgtacagggatag

Forwardagccacatcgctcagacac

Reversegcccaatacgaccaaatcc

LAT4) and 4F2hc. Two different housekeeping genes,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

2.7. Biodistribution Study in Miceand beta actin (ACTB) were used as internal controls to

In vivo experiments were performed to investigate thecompensate for the differences between the initial RNA

biodistribution of 6-I-D-mTyr administered to mice. For

and cDNA amounts. An Mx3005P thermocycler (Agilent

3-5 male ddY mice (6 weeks old) in each group, 0.1 ml ofTechnologies) was used for the qRT-PCR reactions.

saline containing 11.1 kBq of 6-I-D-mTyr was injected

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Basic study on 6-iodo-D-m-Tyr

into the tail vein. Animals were anesthetized with ether

it was decided that the transport system analyses using aand sacrificed by heart puncture at 2, 5, 10, 15, and 30 min

specific transport system inhibitor would be conducted atafter the injection. They were dissected and the brain,

10 min after administration of the labeled compound.

heart, lung, liver, pancreas, spleen, kidney, stomach, and7

sampled. The radioactivity in each organ was determined

6

using a well-type scintillation counter (ARC-300; Aloka).
The biodistribution of the radioactivity was determinedfor each organ as percent dose/organ and on a per-organweight basis as percent dose/g.

Uptake(% of administered dose)

intestines were removed and weighed. Blood was also

5
4
3
2
1
0

2.8. Statistical Analysis
Data were collated as mean ± standard deviation of 3-5

0

10

20

30
40
Time (min)

50

60

70

Fig. 3. Time course of 6-I-D-mTyr uptake.

measurements, and each experiment was performed in
duplicate. Results were analyzed using the Student’s t test.
Probability levels of P<0.0001 were considered to indicate
statistical significance.

3.3. Labeled Amino Acid Uptake Inhibition Study
Inhibition of transport system L was dependent on theconcentration of the inhibitor, and maximum inhibition
was seen at 10 mM (Fig. 4-A). Based on that result, we

3. Results

analyzed transport system L using a BCH concentrationof 10 mM.
Inhibition of transport system A was not dependent on

3.1. Cell Culture and Proliferation Curves
The cells showed continuous logarithmic growth

the concentration of the inhibitor. There were somebeginning on Day 1 after seeding into the culture dishes

concentrations at which increased uptake was observed,(Fig. 2), and confluent growth was seen on Day 4. The

but there were no statistically significant differences (Fig.

number of cells decreased thereafter. Therefore, the cells

4-B). We analyzed transport system A at the MeAIB

were used in experiments on Day 3 after seeding.

concentration that showed the greatest inhibitory effect, 1mM.
The contribution of transport system L to the uptake of
6-I-D-mTyr was 54.0±1.0%, which was statistically

3.2. Time Course of 6-125I-D-mTyr Uptake
When the dishes were incubated for 10 min, 6-I-D-

significant (P<0.001). The contribution of transport

mTyr uptake was 5.58±0.35%, and thereafter, 6-I-D-mTyrsystem A to 6-I-D-mTyr uptake was 6.0±6.0%, which was

was gradually washed out of the cells (Fig. 3). As a result,

not statistically significant (P=0.561) (Fig. 4-C). The

107

contribution of transport system L to the uptake of [14C(U)]

Cell count (No./dish)D-tyrosine was 70.0±6.0%, which was statisticallysignificant (P<0.001). The contribution of transportsystem A to [14C(U)]D-tyrosine uptake was 3.0±3.1%,

106

which was not statistically significant (Fig. 5). The uptakeof both [14C(U)]D-tyrosine and 6-I-D-mTyr occurred via
similar transport systems.

105

0

1

2

3

4

Time (days)

Fig. 2. Growth curve of DLD-1 cells.

5

3.4. qRT-PCR Analysis of DLD-1 Cells
The expression of amino acid transport system genes in

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茨城県立医療大学紀要

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PBS (Na+(+))

120

PBS (Na+(−))

100
80

120

60
40
20
0
0

1

2

3

4

5

6

7

8

9

10

D -[

14

The concentration of BCH [mM]

C(U)]tyrosine %uptake [relative value]%uptake [relative value]A

B%uptake [relative value]

第 24 巻

100
80
60
40

*

20

*

0

160
140
120
100
80
60
40
20
0

control

MeAIB(1 mM)

Na+(-)BCH(1 mM)

4℃

Fig. 5. The effect of inhibitors (MeAIB, BCH) on D-[14C(U)]tyrosine uptake into DLD-1 cells incubated at 4°C in Na+free medium. Values represent mean ± SD as percent ofadministered dose (n = 4). *; P<0.0001 (statistical significant).

0

1

2

3

4

5

6

7

8

9

10

transport system, system L, and the coupling factor 4F2hc

The concentration of MeAIB [mM]Cgene (Fig. 6). Transporters of the Na+-independent

PBS (Na+(+))

were highly expressed in DLD-1 cells.

PBS (Na+(−))

copies/1uL of cDNA
0

6-I-D-m Tyr %uptake [relative value]120

500,000

1,000,000

1,500,000

2,000,000

4F2hc

100
LAT1

80

*

60
40

LAT2

*
LAT3

20
0
control

MeAIB(1 mM)

Na+(-)

BCH(10 mM)

4℃

Fig. 4 (A) Curve demonstrating concentration-dependent
inhibition of 6-I-D-mTyr uptake by BCH.
(B) Curve demonstrating concentration-dependent inhibition of
6-I-D-mTyr uptake by MeAIB.
(C) The effect of inhibitors (MeAIB, BCH) on 6-I-D-mTyruptake into DLD-1 cells incubated at 4°C in Na+-free medium.
Values represent mean ± SD as percent of administered dose (n= 4). *; P<0.0001 (statistical significant).

LAT4

Fig. 6. Results of real-time PCR analyses.

3.5. Biodistribution Study in Mice
Following iv administration in normal mice, 6-I-DmTyr rapidly moved out of the blood and into the tissues(Table 2 A, B). The rapid decrease in the level of 6-I-DmTyr in the blood and conspicuous accumulation in the

DLD-1 cells was analyzed using qRT-PCR. The hightumor tissues on imaging support its usefulness as aquality of the total RNA harvested from each tumor cell

molecular imaging agent for tumors. Also, accumulation

was confirmed using a bioanalyzer (data not shown). The

in the pancreas showed the characteristic in vivo kinetics

expression levels of neutral amino acid transport system

of AAs. Almost no radioactivity accumulation was seen in

genes were calculated using a calibration curve for eachthe stomach, indicating high resistance of 6-I-D-mTyr to

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Basic study on 6-iodo-D-m-Tyr

Table 2. Biodistribution of 6-I-D-mTyr in normal mice.

A Biodistribution of 6-I-D-mTyr in mice (% dose/g)
2 min

5 min

10 min15 min30 minBlood

6.531±0.416

3.605±0.415 2.327±0.222 1.491±0.102 0.675±0.160

Brain

0.218±0.012

0.151±0.065 0.081±0.012 0.067±0.013 0.050±0.013

Pancreas4.528±0.298

3.594±0.304 2.368±0.274 1.977±0.648 0.594±0.151

Spleen2.228±0.182

1.371±0.149 0.964±0.218 0.702±0.117 0.330±0.088

Stomach0.441±0.140

0.342±0.074 0.441±0.205 0.512±0.225 0.564±0.054

Intestine

1.687±0.151

1.082±0.223 0.641±0.063 0.533±0.061 0.411±0.038

Liver

7.984±1.078

6.416±0.397 5.066±0.557 4.582±0.881 2.387±0.245

Kidney45.321±10.681 18.706±0.432 9.770±1.243 18.543±0.462 5.274±5.037

Heart

2.874±0.226

1.372±0.340 0.828±0.078 0.770±0.143 0.365±0.097

Lung

4.349±0.331

2.391±0.248 1.565±0.207 1.413±0.204 0.667±0.113

All values are mean ± SD.
B Biodistribution of 6-I-D-mTyr in mice (% dose/organ)
2 min

5 min

10 min15 min30 minBlood2.613±0.166 1.442±0.166 0.931±0.089 0.596±0.041

0.270±0.064

Brain

0.075±0.011 0.055±0.023 0.032±0.005 0.026±0.004

0.020±0.005

Pancreas 0.586±0.150 0.535±0.093 0.364±0.076 0.254±0.060

0.078±0.008

Spleen0.159±0.012 0.121±0.036 0.097±0.045 0.056±0.016

0.032±0.010

Stomach 0.534±0.059 0.325±0.043 0.351±0.143 0.389±0.122

0.437±0.161

Intestine 3.727±2.227 3.046±0.540 1.770±0.175 1.409±0.124

1.090±0.151

Liver

12.090±1.827 10.096±0.937 7.970±0.927 6.482±0.810

3.627±0.402

Kidney 15.472±3.392 7.155±0.524 3.543±0.845 6.671±0.597

1.905±1.851

Heart

0.343±0.057 0.161±0.063 0.082±0.005 0.076±0.021

0.036±0.008

Lung0.689±0.067 0.423±0.063 0.325±0.139 0.261±0.019

0.136±0.022

All values are mean ± SD.

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茨城県立医療大学紀要

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de-iodination. This suggests that it will be possible to

第 24 巻

predominantly occurs via membrane transport.

avoid undesirable radiation exposure of the thyroid,

As the membrane transport responsible for thestomach, and other organs due to the release of radioactive

accumulation of 6-I-D-mTyr involves an AA transport

iodine via de-iodination reactions. There was somesystem, we then carried out uptake inhibition experiments.

accumulation in the intestine and liver on a per-organ

Various AA membrane transport systems have beenbasis, but on the per-organ-weight basis this accumulation

described, including transport system L, transport system

was minor. Physiologic accumulation in normal mouseA, and transport system ASC, among others. Transport

tissues was low overall, and since there was little

systems A and ASC depend on the presence of Na+, andphysiologic accumulation in the brain and lungs, 6-I-D-

they can be distinguished on the basis of specific inhibition

mTyr may be effective for tumors in these organs.

by artificial AAs and on pH dependence. That is, transportsystem A is Na+ dependent, and uptake is inhibited by theartificial AA MeAIB. Like transport system A, transport

4. Discussion

system ASC is Na+ dependent, but it is not inhibited by
MeAIB. Transport system L is independent of Na+, and it

In our research into 6-I-D-mTyr, we first took
into

consideration

measurementofis specifically inhibited by a different artificial AA, BCH.

L-3,4-

In our present experiments, we examined 6-I-D-mTyrdihydroxyphenylalanine (DOPA) decarboxylase activity

uptake into DLD-1 cells to determine what kind ofand developed an L-isoform (6-I-l-mTyr) as a cranial

dependence it showed in terms of the concentration of

nerve transmission function imaging agent for cerebral

MeAIB as a specific inhibitor of transport system A, or the

dopaminergic presynaptic function.16) Based on the results

concentration of BCH a specific inhibitor of transportof rat brain experiments using inhibitors, we hypothesized

system L. Our aim was to determine the concentration ofthat 6-I-l-mTyr would be useful as a diagnostic imaging

each of those compounds to be used in inhibition

agent that reflects both the activity of AA transport

experiments. We found that 6-I-D-mTyr uptake into DLD-

enzymes (for which an inhibitory effect was seen) and the

1 cells did not show any clear dependence on thelevel of DOPA decarboxylase activity. On the other hand,concentration of MeAIB, so we decided to use thisalthough the optical isomer of 6-I-l-mTyr, 6-I-D-mTyr,inhibitor at 1 mM in the inhibition experiments (Fig. 4-B).

reflected AA transport in the same rat brain studies, weConversely, the uptake of 6-I-D-mTyr into DLD-1 cells

found that it did not reflect the level of DOPA decarboxylase

was dependent on the concentration of BCH, and theactivity. It was thus concluded that the D-isomer could notamount of 6-I-D-mTyr taken up decreased as the BCH

be used to quantify DOPA decarboxylase activity.

concentration increased. We therefore decided on aWith the objective of identifying a different applicationfor 6-I-D-mTyr, in the present study, we basicallyconcentration of 10 mM of this inhibitor for the uptake
inhibition experiments (Fig. 4-A).

examined its potential as a diagnostic imaging agent for

Uptake of 6-I-D-mTyr did not differ significantly as atumors in which AA transport is said to be enhanced. First,

function of whether or not Na+ was included in the PBS,

we generated curves for 6-I-D-mTyr uptake into humanleading us to conclude that neither of the Na+-dependentcolon tumor DLD-1 cells over time, thereby confirming

transport systems (i.e., systems A and ASC) is involved inthat this uptake occurs. When 4°C PBS containing Na+

6-I-D-mTyr uptake by DLD-1 cells. It became clear thatwas used as the uptake solution, 6-I-D-mTyr was

the hypothesis that transport system L is primarily

incorporated into DLD-1 cells, and that accumulation was

responsible for 6-I-D-mTyr uptake is correct (Fig. 4-C).

reduced by 71.0±5.0% in the presence of a membrane

Thus, there was no contradiction with real-time PCR

transport inhibitor. About 30% of the total uptake wasresults demonstrating expression of the LAT1, LAT2,

estimated to be due to diffusive transport. Accordingly, we

LAT3, and LAT4 transporter genes of transport system L

confirmed that 6-I-D-mTyr accumulation in DLD-1 cells

and the 4F2hc coupling factor. Cellular accumulation ofPresented by Medical*Online

9

Basic study on 6-iodo-D-m-Tyr

D-[14C(U)]tyrosine can also be thought of as primarily

byadministering

6-I-D-mTyr

to

mice

harboring

due to transport system L, and a similar discussion applies

transplanted tumors, followed by imaging for visual(Fig. 5).

verification of the biodistribution.

Among the isoforms of transport system L AA
transporters, the LAT1 and 4F2hc heterodimers have
5. Conclusion

received considerable attention in regard to tumors andactivation of leukocytes.

20)

It is hoped that there will be

studies of 6-I-D-mTyr transport and the expression of

We confirmed that DLD-1 cells accumulate 6-I-D-mTyrtumor-related AA transport systems other than the ones

via transport system L. Experiments in mice confirmed

we examined in the present study, such as B , ASCT2,

that 6-I-D-mTyr is highly resistant to de-iodination.

and xCT.

Moreover, the in vivo accumulation of 6-I-D-mTyr in

0,+

21)

We also investigated the biodistribution of 6-I-D-mTyr.

normal mouse tissues is low. Following administration to

Following iv administration to normal mice, 6-I-D-mTyrnormal mice, the concentration of 6-I-D-mTyr in various

moved out of the blood and into the tissues and wasorgans and tissues decreased rapidly. In contrast, 6-I-D-

rapidly eliminated from the blood (Table 2 A, B). The

mTyr that had accumulated inside tumor cells exhibiting

rapid decrease in the level of 6-I-D-mTyr in the blood andenhanced expression of transport system L transporters

conspicuous accumulation in the tumor tissues on imaging

was retained for a long time. In this situation, 6-I-D-mTyrsupport its usefulness as a molecular imaging agent forhas the potential to be highly useful as a newtumors. Also, the accumulation of 6-I-D-mTyr in theradiopharmaceutical for molecular imaging of tumors.

pancreas showed the characteristic in vivo kinetics ofFurther investigation is expected for molecular imaging

AAs. Almost no radioactivity accumulation was seen inof tumors via SPECT using 6-I-D-mTyr.

the stomach, indicating high resistance of 6-I-D-mTyr tode-iodination. This suggests that it will be possible toConflict of Interest

avoid undesirable radiation exposure of the thyroid,
stomach, and other organs due to radioactive iodine ( I125

I-) released by de-iodination reactions. There was someaccumulation in the intestine and liver on a per-organ

The authors declare that they have no conflict ofinterest.

basis, but on the per-organ-weight basis it was minor.
Physiologic accumulation in normal mouse tissues was
Acknowledgement

low overall, and since there was little physiologic
accumulation in the brain and lungs, 6-I-D-mTyr may be

We wish to thank Yuko Hamai, Sanae Matsutani, Naomi

effective for evaluating tumors in these organs.
In general, many tyrosine analogues show high levels

Tojyo, Yumi Suzuki, Yuko Hirotsu, Sayaka Imai, and Keiof excretion in the urine, and it is therefore said that they

Satoh (Ibaraki Prefectural University of Health Sciences)

are unsuitable for visualizing organs that are in the vicinity

for their excellent technical assistance. This work wasof the kidneys and urinary tract.17) The accumulation rate

supported by Grants-in-Aid for Scientific Researchof tyrosine analogues in the kidney is low in comparison(#26461801) from the Ministry of Education, Science,with 125I-3-alpha-methyl tyrosine (IMT).17) It has beenSports and Culture of Japan and the Japan Society for thesuggested that, at the time of testing, it is relatively

Promotion of Science. Financial support was also provided

advantageous to have the patient urinate as a pretreatment

by a Japan Atherosclerosis Research Foundation Grant into eliminate radioactivity from the urinary tract, as this2016-2018.

will improve imaging of tumors in the colon and otheradjacent organs. It is hoped that studies will be carried out

Presented by Medical*Online

茨城県立医療大学紀要

10

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和文抄録
123

I-6-iodo-D-meta-tyrosine（123I-6-D-mTyr）は，腫瘍分子イメージング用の放射性ヨウ素標識 D- アミノ酸

候補化合物である。その 125I 標識体を用い DLD-1 ヒト大腸癌株化細胞における集積のメカニズムと正常マウ
スにおける体内分布を検討した。DLD-1 による検討では，分子デザインの母体化合物 D- [14C(U)] tyrosine を比較のために用いた。双方の D 体アミノ酸は，約 50％が中性アミノ酸輸送系 L によるものであった。正常マウスの組織における 125I-6-D-mTyr の生理学的集積は全体的に低く，速やかな血液からのクリアランスが観察された。癌細胞とマウスによるこれらの検討から，腫瘍分子イメージングにおける新しい診断薬としての
125

I-6-D-mTyr の可能性が示唆された。

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